Annals of the Royal College of Surgeons of England (I979) vol 6i

Bile studies after liver transplantation Paul McMaster FRCS Consultant Surgeon, University Department of Surgery, Addenbrooke's Hospital, Cambridge

Summary An analysis of bile composition following orthotopic liver transplantation in the rhesus monkey showed that during rejection only small quantities of viscid bile were produced and that this was associated with increased cholesterol saturation. Bile composition in patients after liver transplantation also showed that bile was supersaturated with cholesterol in the early postoperative period while the enterohepatic circulation of bile acids was interrupted by a draining T tube. However, further study of patients showed a poor correlation between bile composition and the development of biliary complications. An analysis of bile 'sludge' showed that after transplantation two types were encountered. The first, containing large quantities of unconjugated bilirubin, was present when intrabiliary obstruction was associated with long-standing mechanical obstruction. The second type, present in patients developing masses of intrabiliary 'sludging' shortly after transplantation, consisted mainly of necrotic donor biliary tract due to damage during preservation. An intrabiliary perfusion technique was developed which in animal models reduced the extent of donor biliary damage. Introduction There has recently been a significant improvement in the overall results of orthotopic liver transplantation, and an increasing number of patients are being treated successfully",2. Nevertheless, biliary complications still occur after transplantation, and a preliminary analysis of the Cambridge/King's College Hospital series of patients showed that I 9 of 31, patients developed major biliary complications, and in I 2 this led directly to the patient's death. The most commonly encountered biliary complication was the development of a biliary fistula due to anastomotic breakdown, mech-

anical obstruction, and cholangitis, and in I4 of these i 9 patients there was majpor biliary duct obstruction by masses of intMaluminal biliary 'sludge'. The pathogenesis of this common disorder is unknown. A clearer understanding of the blood supply of the biliary tract3 and improved biliary anastomotic techniques4 have resulted in a dramatic reduction in the incidence of biliary leakage, and of the last 32 patients undergoing orthotopic transplantation, only 3 have developed significant biliary fistulae. In addition, the gallbladder conduit technique has meant that few mechanical biliary obstructions have been encountered, although a high proportion of patients still develop intraluminal obstruction with biliary 'sludging' and cholangitis (Fig. i). Preliminary analysis suggested that biliary 'sludge' consists of bile constituents5'6, and ..

FIG. I Massive intrabiliary 'sludging', with fulminating cholangitis in the absence of mechanical obstruction, encountered within 6 weeks of orthotopic transplantation. Arris and Gale Lecture delivered at King's College Hospital, London, on i9th December I9,78

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Paul McAMaster undertaken to clarify these changes and to evaluate their postoperative importance.

Animal studies ANIMAL MODEL

A model of orthotopic liver transplantation was developed in the rhesus monkey (Macaca mulatta) which incorporated the extracorporeal bile circuit, enabling small samples of bile to be taken continuously (Fig. 2)10. A preliminary study in non-transplanted rhesus monkeys had not shown any reduction in bile flow or alteration in bile constituents with standard immunosuppression (azathioprine 2.5 mg/kg body weight and prednisolone I.5 mg/kg), nor had temporary total hepatic ischaemia produced bile changes"1. TRANSPLANTATION

FIG. 2 Basic bile circuit enabling continuous

5% diversion of bile with return of the remainder to the enterohepatic circulation, allowing maintenance of biliary composition. animal experimental studies have suggested an alteration in bile composition following liver transplantation7-9. However, the precise characteristics of bile composition after liver transplantation are uncertain and their importance in the development of biliary complications is unclear. We considered it quite possible that damage to the donor liver either by ischaemic damage during harvesting and storage or rejection or damage due to hepatotoxic drugs might lead to reduction in the bile acid content of the bile, with supersaturation and subsequent cholesterol precipitation. Bile studies after liver transplantation were 1000+

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BILE ANALYSIS

Bile analysis was carried out with standard biliary analytical techniques and the theoretical cholesterol saturation index calculated"2. In addition, total acid pool and bile synthesis p SGPT

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MODEL

Donor and recipient animals were matched for blood group and weight (2-5 kg). The donor technique consisted of full skeletonisation of the donor liver and isolation, with intraportal perfusion with 250 ml of Hartmann's solution at 40C followed by IOO ml of plasma protein fraction (PPF) at 40C. Donor livers were then stored in ice-cold saline at 40C while the recipient animal was prepared. In the recipient animal a total hepatectomy was performed and an orthotopic hepatic transplantation carried out. Direct end-to-end caval anastomosis was performed, the donor hepatic artery being transplanted directly into the recipient aorta.

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FIG. 3 Fall in daily bile volume (ml) as rejection occurs with deteriorating liver function in one animal. (Biliary bilirubin measured in mg/Ioo ml (=I7.I ,umol/l); alkaline phosphatase and SGPT in U/l).

Bile studies after liver transplantation were measured after complete interruption of the enterohepatic circulation" '. Statistical analysis was carried out by computer analysis of variants for individual animals and groups, with log transformation when handling proportions.

RESULTS

Orthotopic liver transplantation was initially carried out and bile studies were possible in 4 animals transplanted without immunosuppression. Bile flow in the first 4 or 5 days was satisfactory, but after this time, as rejection occurred with rapid deterioration of liver function, the bile volume fell (Fig. 3). The fall in bile volume was accompanied by a reduction in total bile acid output and a statistically significant increase in total bile viscosity. After 7 days there was no further

bile output as liver function deteriorated further. However, in the first 5 days before major rejection occurred the composition and flow of bile were not significantly different from those in control animals or immunosuppressed animals. A further 5 animals were transplanted and in addition received immunosuppression with azathioprine 2.5 mg/kg and prednisolone I.5 mg/kg, and studies of bile composition and flow were carried out for 25, 29, 33, 22, and The main changes in 54 days respectively. are summarised in and composition flow bile the table, the bile being analysed in three phases: the early phase between days 2 and 5 after transplantation; the intermediate phase between days 7 and I4; and the late phase between days 20 and 30. The table clearly shows the significant re-

Changes in bile fow and composition following orthotopic liver transplantation rhesus monkeys

in

Phase

(ml)

61.0 ± 42.1 2.25 ± 2.1

Bile viscosity

1.83 ± 1.2

(mmol/day)

(CP) Bile acid concentration

**

147.8 ± 11.2

017.1 ± 30.1

(n=22)

(n=40)

(n=20)

Bile acid output

Late (20-30 days)

Intermediate (7-14 days)

Early (2-5 days) Daily bile volume

**

0.74

**

5.79

7.70 ± 0.^

1.4

(n=23)

(n=40)

(n=20)

1.09 ± 0.08

1.93

(n=22)

(n=13)

(n=19) 35.21 ± 10.6

40.55 ± 7.7

6.25 ± 1.2

5.98 ± 1.6

1.82 ± 0.7

2.16 ± 0.5

*

52.11 ± 4.6

(mmol/l) Phospholipid concentration

6.19 ± 0.9

(mmol/l) Cholesterol concentration

*

1.16 ± 0.3

(mmol/l) Bile Constituents Bile acid Phospholipid Cholesterol Cholesterol saturation index

79.81 14.42 3.91 77.44

437

**

82.40 11.93 4.50 94.38

87.53 10.36 1.82 44.62

Mean values are shown ± 1 SD * P < 0.05 ** P< 0.01 Bile acid (as taurocholic acid): 1 mmol = 515 mg; 1 mmol/l = 51.5 mg/100 ml. Phospholipid: 1 mmol/l = 0.774 g/l. Cholesterol: 1 mmol/l - 38.67 mg/100 ml.

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Paul McMaster

duction in bile volume and bile acid output and while the supersaturation of bile in this in the intermediate phase during rejection, early phase may be significant in the prowith a high bile viscosity. This is associated duction of biliary 'sludge', it seemed unlikely with a relative reduction in bile acid con- that this was the only factor. centration compared with control intermediate 'SLUDGE ANALYSIS animals, with the maintenance of biliary BILE 'sludge' obtained at operSamples cholesterol concentrations, so that the overall ation or ofat biliary were analysed both postmortem cholesterol saturation index rises. Although TRANS PLANT the mean cholesterol saturation index falls below the theoretical maximum of io07o, in inT Tube closed 60 dividual animals bile was frequently supersaturated with cholesterol. As animals recovered from the rejection episode bile composi50 Acid tion returned to 'normal' by the late phase. The raised cholesterol saturation was also accompanied by a raised mean biliary bilirubin 40 I1

level. Thus severe rejection encountered in the rhesus monkey resulted in small quantities of extremely viscid bile with a raised cholesterol saturation index and increased biliary bilirubin content (Fig. 3). Human studies Although major rejection episodes were encountered in all rhesus monkeys, significant rejection in man following liver transplantation is uncommon'3 and studies were therefore undertaken in man to see if bile changes did occur following transplantation. Analysis of bile draining via the biliary T tube was carried out and it was soon clear that the major factor in determining bile composition was the interruption of the enterohepatic circulation of bile acids due to the draining T tube. Indeed, as soon as the T tube was closed, with re-establishment of the normal enterohepatic circulation of bile acids, biliary bile acid and phospholipid levels rose, with a significant reduction in cholesterol saturation from its supersaturated levels (Fig. 414), although the bile remained intermittently supersaturated with cholesterol and massive biliary 'sludging' was encountered. In 7 patients bile flow was sufficiently consistent in the postoperative period to allow analysis to be undertaken. In 4 patients significant biliary complications occurred, but in 3 no problems were encountered. However, a comparison between the bile constituents and cholesterol saturation between those who developed complications and those who did not failed to show any significant difference,

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Days TRANS PLANT

Rejection Treatment

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FIG. 4 Changing patterns in human bile chemistry following liver transplantation showing marked change with closure of T tube and re-establishment of enterohepatic circulation. (Reproduced by permission from Transplant-

ation'4.)

Bile studies after liver transplantatton

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storage of the biliary tract has received little attention in the literature, and further experimental studies were undertaken to assess the extent of this damage.

FIG. 5 Biliary 'sludge' removed from a patient 8 weeks after transplantation showing a major fibrous organic matrix which, histologically, shows features of necrotic biliary tract. (Haematoxylin and eosin.) biochemically and histologically and it was soon clear that two separate kinds of biliary 'sludge' were being encountered. Type I was present in 6 patients presenting with mechanical obstruction and biliary 'sludging' 6 months to 5 years after liver transplantation. Biochemical analysis of biliary 'sludge' in these patients showed a major constituent to be unconjugated bilirubin. However, in I2 samples of biliary 'sludge' found in patients between 3 and 8 weeks after transplantation the major constituent consisted of a fibrous matrix of organised collagen tissue with relatively small quantities of standard bile constituents. This Type II 'sludge' histologically showed all the features of necrotic donor biliary tract (Fig. 5). Damage to th3i donor biliary tract could occur at varying intervals after orthotopic liver transplantation as a result of ischaemia due to damage of the vascular supply to the biliary tract3 or due to severe infection or poss-

ibly rejection. However, histological studies of the biliary tract taken at the end of preservation of the donor liver showed significant epithelial and bile duct wall damage as a result of storage (Fig. 6). The possibility therefore existed of primary ischaemic damage during the period of storage of the donor liver resulting in necrosis of epithelium and underlying bile duct connective tissue leading to shedding, with intraluminal obstruction. The result of warm and cold ischaemic

Animal studies during liver and biliary system storage A preliminary study in the pig had suggested that a limited period of warm ischaemia did not produce fundamental damage to the biliary tract but that more prolonged periods of cold ischaemia in the presence of bile produced extensive epithelial damage&'. Studies were therefore undertaken in a series of rabbits in which standard liver and biliary preservation was achieved using the human donor liver perfusion technique with Hartmann's solution and PPF, and it became clear that cold storage of the liver and biliary tract after standard perfusion for periods of longer than i h in the presence of bile resulted in severe autolytic damage to the donor biliary tract. After 6 h of storage there was extensive necrosis of the bilary tract with damage to adjacent hepatic tissue. In an attempt to improve the preservation of the biliary tract during cold storage a further series of animals was studied in which, in addition to the standard intraportal infusion, bile from the gallbladder and common bile duct was washed out and the biliary tract perfused with PPF at 40C. With a can-

FIG. 6 Histology at the end of 243 minutes' preservation (O initial warm ischaemia, 26 minutes ischaemia during revascularisation) showing loss of epithelium with subendothelial autolysis and pigment infiltration into the underlying connective tissue layers. (Haematoxylin and eosin.)

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Paul McMaster

bladder 50-100 ml of PPF was perfused I gratefully acknowledge the support of my colleagues Cambridge and at King's College Hospital nula introduced into the apex of the gall- in this study, and particularly Dr Richard throughout through the gallbladder, cystic duct, and bili- Waldram, Dr Roger Williams, and Professor R Y ary tract with, in addition, flushing out of the Calne. In addition, Dr Theodore Syrakos and Dr common bile duct and common hepatic duct. Derek Wight were involved in the later animal Both light and electron microscopic studies of studies of biliary duct preservation. the biliary tract carried out at 6 and I 2 h revealed excellent preservation of the biliary References tract with maintenance of normal epithelial IStarzl, T E, Porter, K A, Putnam, S W, Schroter, G P J, Halgrimson, C G, Weil, R III, Hoielscher, architecture and without evidence of underlying M, and Reid, H A S (1976) Surgery, Gynecology autolysis or damage to the bile duct and galland Obstetrics, 142, 487. bladder walls. This technique is currently be- 2 Calne, R Y, and Williams, R (I977) British ing evaluated in the clinical human transMedical Journal, I, 471. plantation programme prior to hepatic and 3 Northover, J, and Terblanche, J (I978) Transplantation, 26, 67. biliary storage.

Conclusions Orthotopic liver transplantation in man remains a major operation olften undertaken in patients who are critically ill. In the past a number of difficulties have been encountered, but of late the major difficulty has sur-

4 Calne, R Y (I976) Annals of Surgery, I84, 605. 5 Waldram, R, Williams, R, and Calne, R Y (I975) Transplantation, I9, 382. 6 Starzl, T E, Putnam, C W, Hansbrough, J F, Porter, K A, and Reid, H A S (1977) Surgery, 81, 212. 7 Martineau, G, Porter, K A, Corman, J, Launois, B, Schroter, G T, Palmer, W, Putman, C W, Groth, C G, Halgrimson, C G, Penn, I, and Starzl, T E (1972) Surgery, 72, 604. 8 Bell, P, Homatas, J, MacSween, R, and Brettschneider, L (I969) Surgical Forum, 20, 295. 9 Daloze, P, Fourtanier, G, Beaudouin, M, Corman, J, Smeesters, C, and Saric, J (I977) Transplantation Proceedings, 9, 309. io Dowling, R H, Mack, E, and Small, D M (I970) Journal of Clinical Investigation, 49, 232. ii McMaster, P, Walton, R M, Wight, D G D, Medd, R K, and Syrakos, T P, British Journal of Surgery. In press. 12 Holzbach, R T, Marsh, M, Olszewski, M, and Holan, K (I973) Journal of Clinical Investigation, 52, 1467. I3 Calne, R Y (I977) Transplantation Proceedings,

rounded the Achilles heel of the operationnamely, the biliary tract. The development of newer biliary anastomotic techniques has reduced significantly the incidence of biliary fistula formation, and these aniimal and human studies have clarified the changes in bile composition and flow following orthotopic liver transplantation. They would suggest that in the majority of patients developing intrabiliary obstruction due to biliary 'sludge' the mechanism consists of damage to the donor biliary tract during the period of cold storage before transplantation. The simple technique of perfusion of the gallbladder and biliary tract with PPF has shown excellent histological 9, 209. appearances after I2 h of storage in animals I4 McMaster, P, Herbertson, B, Cusick, C, Calne, and may well improve the results of human R Y, and Williams, R (I978) Transplantation,

transplantation.

25, 56.